Studies have been initiated to examine the pathway of secretion of pertussis (PT) from B. pertussis. Several mutants which lack the ability to secrete pertussis toxin have been isolated and mutations mapped to a locus directly downstream of the PT structural genes. These mutants were found to be defective in the secretion of PT. This locus was termed the PTL locus for pertussis toxin liberation. The region of the B. pertussis chromosome which appeared to be important for secretion of PT as indicated by mutational analysis was sequenced. Sequence analysis revealed eight open reading frames (ORFs). A search of the Swiss-Prot data base for proteins homologous to be proteins predicted by these ORFs B, C, D, E, F, G, and H were found to be homologous to the VirB3, VirB4, VirB6, VirB8, VirB10, and VirB11 proteins, respectively, from the plant pathogen, Agrobacterium tumefacient. The VirB proteins have been implicated in the transfer of a piece of DNA, T-DNA, across bacterial membranes. After release from the bacterial cell, the T-DNA ultimately crosses plant cell membranes, integrates into the plant cell genome, and codes for biosynthesis of plant growth hormones. Overexpression of these hormones leads to a loss of division control and tumors. Recombinant forms of the proteins predicted by ORFs E, F and G were made in Escherichia coli, isolated and injected into mice to induce antibodies specific for these proteins. Antisera were collected and used to probe extracts of B. pertussis for PtIE, PtIF, and PtIG. The antisera recognized proteins of the expected molecular weights confirming the existence of these proteins. Our data suggest that several accessory proteins may be involved in secretion of PT from B. pertussis and that this transport system may be a member of a family of transport systems found in other types of bacteria.